USPTO Patents Application 10687850

per

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 4 ;

C12Q 1/00, 1/26, C12M 1/34
G01N 27/26

Al

(11) International Publication Number:

WO 89/ 08713

(43) International Publication Date:

21 September 1989 (21.09.89)

(21) International Application Number: PCT/US89/01057

(22) International Filing Date: 14 March 1989 (14.03.89)

(31) Priority Application Numbers:

(32) Priority Dates:

(33) Priority Country:

168,295
Not furnished

15 March 1988 (15.03.88)
3.89)

13 March 1989 (13.03.1

US

(71) Applicant: LIFE-CHEK LABORATORIES [US/US];

4900 Perry Highway, Pittsburgh, PA 15229 (US).

(72) Inventors: POTTGEN, Paul, A. ; 2335 Big Rock Road,

Allison Park, PA 15101 (US). SZUMINSKY, Neil, J. ;
1427 Hillsdale Avenue, Pittsburgh, PA 15216 (US).
TALBOTT, Jonathan, L. ; 165 Woodhaven Drive,
Mars, PA 16046 (US). JORDAN, Joseph ; 1007 Glenn
Circle North, State College, PA 16803 (US).

(74) Agent: YAHWAK, George, M.; Yahwak & Associates,
25 Skytop Drive, Trumbull, CT 0661 1 (US).

(81) Designated States: AT (European patent), AU, BE (Eu-
ropean patent), CH (European patent), DE (Euro-
pean patent), DK, FI, FR (European patent), GB
(European patent), IT (European patent), JP, KR, LU
(European patent), NL (European patent), NO, SE,
SE (European patent), SU.

Published

With international search report.

(54) Title: METHOD AND APPARATUS FOR AMPEROMETRIC DIAGNOSTIC ANALYSIS

(57) Abstract

The present invention relates to a novel method and apparatus for the amperometric determination of an analyte,
and in particular, to an apparatus (10) for amperometric analysis utilizing a novel disposable electro analytical cell (20) for
the quantitative determination of biologically important compounds from body fluids.

FOR THE PURPOSES OF INFORMATION ONLY

C^usedt^ntifyStatespa^
cations under the PCT. s vv

AT Austria

AU Australia

BR Barbados

BE Belgian

B6 Bulgaria

BJ Benin

BR Brazil

CF Central Afiican Republic

CG Congo

CH Switzerland

CM Cameroon

DE Germany, Federal Republic of

DK Denmark

FI Finland

FR Franco

GA Gabon

GB United Kingdom

HU Hungary

IT Italy

JP Japan

KP Democratic People's Republic

of Korea

KR Republic of Korea

IX Ue^tenstein

LK Sri Lanka

LU Luxembourg

MC Monaco

MG Madagascar

ML Mali

MR Mauritania

MW Malawi

NL Netherlands

NO Norway

RO Romania

SD Sudan

SE Sweden

SN Senegal

SU Soviet Union

TD Chad

TG Togo.

US United States of America

WO 89/08713 | PCT/US89/01057

METHOD AND APPARATUS FOR AMPEROMETRIC
DIAGNOSTIC ANALYSIS

The present application is a Continuation- in-Part of our
5 earlier filed application, United States Serial No. 168,295,
filed March 15, 1988.

FIELD OF THE INVENTION:

The present invention relates to a disposable

10 electroanalytical cell and a method and apparatus for
quantitatively determining the presence of biologically
important compounds^such as glucose; TSH; T4; hormones such
as HCG; cardiac glycosides such as Digoxin; antiarrhythmics
such as Lidocaine; antiepileptics such as phenobarbital;

15 antibiotics such as Gentamicin; cholesterol; non-therapeutic
drugs and the like from body fluids.

Although the present invention has broad applications,
for purposes of illustration of the invention specific
emphasis will be placed upon its application in

20 quantitatively determining the presence of two biologically
important compounds — glucose and cholesterol.

WITH RESPECT TO "GLUCOSE;

Diabetes, and specifically diabetes mellitus, is a
25 metabolic disease characterized by deficient insulin
production by the pancreas which results in abnormal levels
of blood glucose. Although this disease afflicts only

WO 89/08713

2 PCT/US89/01057
approximately 4% of the population in the United States, it
is the third leading cause of death following heart disease
and cancer, with proper maintenance of the patient's blood
sugar through daily injections of insulin, and strict
control of dietary intake, the prognosis for diabetics is
excellent. However, the blood glucose levels must be
closely followed in the patient either by clinical
laboratory analysis or by daily analyses which the patient
can conduct using relatively simple, non-technical, methods.

At the. present, current technology for monitoring blood
glucose is based upon visual or instrumental determination
of color change produced by enzymatic reactions on a dry
reagent pad on a small plastic strip. These colorimetric
methods which utilize the natural oxidant of glucose to
gluconic acid, specifically oxygen, are based upon the
reactions:

B-D-Glucose + 0 2 + — > D-Gluconic Acid + H 2 0 2
HjOj + Reagent — Hp + color.

WITH RES PECT TO fTTOLESTTOOT..

Current technology for the determination of cholesterol
is also based upon similar methods.- m the case of
cholesterol, the- methods presently Wed are based upon the
generalized reactions:

Cholesterol + Bp + o 2 — > Cholestenbne +R 2 o z
+ Reagent — > up + color

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3

In all present techniques, Dioxygen is the only direct
oxidant used with the enzyme cholesterol oxidase for the
determination of both free and total cholesterol. Using
conventional test methods, oxygen must diffuse into the
sensor solution during use from the surrounding air in order
to provide sufficient reagent for a complete reaction with
the analyte cholesterol in undiluted serum and whole blood
speciments .

In both instances, the presence of the substance is
determined by quantifying, either colorometrically or
otherwise, the presence of hydrogen peroxide. The present
methods of detection may include direct measurement of the
hydrogen peroxide produced by either spectroscopic or
electrochemical means and indirect methods in which the
hydrogen peroxide is reacted with various, dyes, in the
presence of the enzyme peroxidase, to produce a color that
is monitored.

While relatively easy to use, these tests require
consistent user technique in order to yield reproducible
results. For example, thesie tests require the removal of
blood from a reagent, pad at specified and critical time
intervals. After the time interval, excess blood must be
removed by washing. and blotting, or by blotting alone, since
the color measurement is taken at the top surface of the
reagent pad. Color development is either read immediately
or after a specified time interval.

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4

These steps are dependent upon good and consistent
operating technique requiring strict attention to timing.
Moreover, even utilizing good operating technique,
colorimetric methods for determining glucose, for example,
have been shown to have poor precision and accuracy,
particularly in the hypoglycemic range. Furthermore,
instruments used for the quantitative colorimetric
measurement vary widely in their calibration methods: some
provide no user calibration while others provide secondary
standards.

Because of the general lack of precision and
standardization of the various methods and apparatus
presently available to test for biologically important
compounds in body fluids, some physicians are hesitant to
use such equipment for monitoring levels or dosage. They
are particularly hesitant in recommending such methods for
use by the patients themselves. Accordingly, it is
desirable to have a method and apparatus which will permit
not only physician but patient self -testing of such
compounds with greater reliability.

The present invention addresses the concerns of the
physician by providing enzymatic amperometry methods and
apparatus for monitoring compounds within whole blood,
serum, and other body fluids. Enzymatic amperometry
provides several advantages for controlling or eliminating
operator dependant techniques as well as providing a greater

WO 89/08713 PCT/US89/01057

5

linear dynamic range. A system based on this type of method
could address the concerns of the physician hesitant to
recommend self -testing for his patients.

Enzymatic amperometry methods have been applied to the
laboratory based measurement of a number of analytes
including glucose, blood urea nitrogen, and lactate.
Traditionally the electrodes in these systems consist of
bulk metal wires, cylinders or disks imbedded in an
insulating material. The fabrication process results in
individualistic characteristics for each electrode
necessitating calibration of each sensor. These electrodes
are also too costly for disposable use, necessitating
meticulous attention to electrode maintenance for continued
reliable use. This maintenance is not likely to be
performed properly by untrained personnel (such as
patients), therefore to be successful, an enzyme amperometry
method intended for self-testing (or non-traditional site
testing) must be based on a disposable sensor that can be
produced in a manner that allows it to give reproducible
output from sensor to sensor and at a cost well below that
of traditional electrodes.

The present invention address these requirements by
providing miniaturized disposable electroana lytic sample
cells for precise micro-aliquote sampling, a self-contained,
automatic means for measuring the electrochemical reduction

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of the sample, and a method for using the cell and apparatus
according to the present invention.

The disposable cells according to the present invention
are preferably laminated layers of metallized plastic and
nonconducting material. The metallized layers provide the
working and reference electrodes, the areas of which are
reproducibly defined by the lamination process. An opening
through these layers is designed to provide the sample-
containing area or cell for the precise measurement of the
sample. The insertion of the cell into the apparatus
according to the present invention, automatically initiates
the measurement cycle.

To better understand the process of measurement, a
presently preferred embodiment of the invention is described
which involves a two-step reaction sequence utilizing a
chemical oxidation step using other oxidants than oxygen,
and an electro-chemical reduction step suitable for
quantifying the reaction product of the first step. One
advantage to utilizing an oxidant other than dioxygen for
the direct determination of an analyte is that they may be
prepositioned in the sensor in a large excess of the analyte
and.thus ensure that the oxidant is not the limiting reagent
(with dioxygen, there is normally insufficient oxidant
initially present in the sensor solution for a quantitative
conversion of the analyte).

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7

In the oxidation reaction, a sample containing glucose,
for example, is converted to gluconic acid and a reduction
product of the oxidant. This chemical oxidation reaction
has been found to precede to completion in the presence of
an enzyme, glucose oxidase, which is highly specific for the
substrate B-D-glucose, and catalyzes oxidations with single
and double electron acceptors. It has been found, however,
that the oxidation process does not proceed beyond the
formation of gluconic acid, thus making this reaction
particularly suited for the electrochemical measurement of
glucose.

In a presently preferred embodiment, oxidations with one
electron acceptor using ferricyanide, ferricinium, cobalt
(III) orthophenantroline, and cobalt (III) dipyridyl are
preferred. Benzoquinohe is a two electron acceptor which
also provides excellent electro-oxidation characteristics
for amperometric quantitation.

Amperometric determination of glucose r for example, in
accordance with the present invention utilizes Cottrell
current micro-chronoamperometry in- which glucose plus an
oxidized electron acceptor produces gluconic acid and a
reduced acceptor. This determination involves a preceding -
chemical oxidation step catalyzed by a bi-substrate bi-
product enzymatic mechanism as will become apparent
throughout this specification.

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In this method of quantification, the measurement of a
diffusion controlled current at an accurately specified time
(e.g. 20, 30, or 50 seconds, for example) after the instant
of application of a potential has the applicable equation
f or amperometry at a controlled potential (E = constant) of:

'COTTOELL 'Dt> "°- 5 'c METABOLITE

at fc > ° at t = o

where i denotes current, nF is the number of coulombs per
mole, D is the diffusion coefficient of the reduced form of
the reagent, t is the preset time at which the current is
measured, and c is the concentration of the metabolite.
Measurements by the method according to the present
invention of the current due to the reoxidation of the
acceptors were found to be proportional to the glucose
concentration in the sample.

The method and apparatus of the present invention
permit, in preferred embodiments, direct measurements of
blood glucose, cholesterol and the !ike. Furthermore, the
sample cell according to the present invention, provides the
testing of controlled volumes of blood without premeasuring. -
Insertion of the sampling cell into the apparatus thus
permits automatic functioning and timing of the reaction
allowing for patient self-testing with a very high degree of
precision and accuracy.

WO 89/08713 PCT/US89/01057

9

One of many of the presently preferred embodiments of
the invention for use in measuring B-D glucose is described
in detail to better understand the nature and scope of the
invention. In particular, the method and apparatus
5 according to this embodiment are designed to provide
clinical self -monitoring of blood glucose levels by a
diabetic patient. The sample cell of the invention is used
to control the sampling volume and reaction media and acts
as the electrochemical sensor. In this described

10 embodiment, benzoquinone is used as the electron acceptor.

The basic chemical binary reaction utilized by the
method according to the present invention is:

B-D-glucose + Benzoquinone + — >GluconicAcid + Hydroquinone

Hydroquinone — >benzoquinone + 2e * + 2H+
15 The first reaction is an oxidation reaction which

proceeds to completion in the presence of the enzyme glucose
oxidase. Electrochemical oxidation takes place in the
second part of the reaction and provides the means for
quantifying the amount of hydroquinone produced in the
20 oxidation reaction. This holds true whether catalytic
oxidation is conducted with two-relectron acceptor^ or one
electron acceptors such as ferricyanide [wherein the redox
couple would be Fe(CN) 6 ~ 3 /Fe (CN) 6 ~ 4 ], ferricinium, Cobalt III
tris orthophenantroline and^ cobalt (III) trisdipyridyl .
25 Catalytic oxidation by glucose* oxidase is highly

specific for B-D-glucose, but is nonselective as to the

WO 89/08713

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10

oxidant. it has now been discovered that the preferred
oxidants described above have sufficiently positive
potentials to convert substantially all of the B-D-glucose
to gluconic acid. Furthermore, this system provides a means
by which amounts as small as l mg of glucose (in the
preferred embodiment) to 1000 mg of glucose can be measured
per deciliter of sample - results which have not previously
been obtained using other glucose self-testing systems.

The sensors containing the chemistry to perform the
desired determination, constructed in accordance with the
present Invention, are used with a portable meter for self-
testing systems. m use the sensor is inserted into the
meter which turns the meter on and initiates a wait for the
application of the sample. The meter recognizes sample
application by the sudden charging current flow that occurs
when the electrodes and the overlaying reagent layer are
initially wetted by the sample fluid. Once the sample
application is detected, the meter begins the reaction
incubation step (the length of which is chemistry dependent)
to allow the enzymatic reaction to reach completion. This
period is on the order of 15 to 90 seconds for glucose, with
incubation times of 20 to 45 seconds preferred. Following
the incubation period, the instrument then imposes a known
potential across the electrodes and measures the current at
specific time points during the Cottrell current decay.
Current measurements can be made in the range of 2 to 30

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11

seconds following potential application with measurement
tiroes of 10 to 20 seconds preferred. These current values
are then used to calculate the analyte concentration which
is then displayed. The meter will then wait for either the
user to remove the sensor or for a predetermined period
before shutting itself down.

The present invention provides for a measurement system
that eliminates several of the critical operator dependant
variables that adversely affect the accuracy and reliability
and provides for a greater dynamic range than other self-
testing systems.

These and other advantages of the present invention will
become apparent from a perusal of the following detailed
description of one embodiment presently preferred for
measuring glucose and another for measuring cholesterol
which is to be taken in conjunction with the accompanying
drawings in which like numerals indicate like components and
in which:

FIG. 1 is an exploded view of a portable testing
apparatus according to' the present invention;

FIG. 2 is a plan view of the sampling, cell of the
present invention;

FIG. 3 is an exploded view of the sample cell shown in
Figure 2;

FIG. 4 is an exploded view of another embodiment of a
sample cell according to the invention;

10

WO 89/08713

PCT/US89/01057

PIG. 5 is a plan view of the cell shown in Figure 4;

FIG. 6 is still another embodiment of a sample cell;

FIG. 7 is a graph showing current as a function of
glucose concentration;

FIG. 8 is a graphical presentation of Cottrell current
as a function of glucose concentration; and

FIG. 9 is a presently preferred block diagram of an
electrical circuit for use in the apparatus shown in Figure
1.

FIG. 10 is a preferred embodiment of the electrochemical
cell.

With specific reference to Figure i, a portable
electrochemical testing apparatus 10 is shown for use in
patient self -testing, such as, for example, for blood
glucose levels. Apparatus 10 comprises a front and back
housing 11 and 12, respectively, a front panel. 13 and a
circuit board 15. Front panel 13 includes graphic display
panels 16 for providing information and instructions to the
patient, and direct read-out of the test results. While a
start button 18 is provided to initiate an analysis, it is

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13

preferred that the system begin operation when a sample cell
20 is inserted into the window 19 of the apparatus.

With reference to Figure 3, sample cell 20 is a
metallized plastic substrate having a specifically-sized
opening 21 which defines a volumetric well 21 , when the cell
is assembled, for containing a reagent pad and the blood to
be analyzed. Cell 20 comprises a first 22 and second 23
substrate which may be preferable made from styrene or other
substantially non-conducting plastic. Positioned on second
substrate 23 is reference electrode 24. Reference electrode
24 may be preferably manufactured, for example, by vapor
depositing the electrode onto a substrate made from a
material such as the polyimide Kapton. In the preferred
embodiment, reference electrode 24. is a silver-silver
chloride electrode. This electrode can be produced by first
depositing a silver layer silver chloride by either chemical
or electrochemical means before the substrate is used to
construct the cells. The silver chloride layer may even be
generated in-situ on a silver electrode when the reagent
layer contains certain of the oxidants , such as
ferricyanide, and chloride as shown in the following
reactions :

Ag + Ox — > ag*+ Red

Ag* + CI" > AgCl

Alternatively th& silver-silver chloride electrode can be
produced by depositing a layer of silver oxide (by reactive

WO 89/08713 „™ t „

PCT/US89/01057

10

15

20

25

14

sputtering) onto the silver film. This silver oxide layer
is then converted in-situ at the time of testing to silver
chloride according to the reaction:

Ag z O + HjjO + 2C1* > 2AgCl + 2 (OH) "

when the sensor is wetted by the sample fluid and
reconstitutes the chloride containing reagent layer. The
silver electrode with a layer containing silver chloride.

The reference electrode may also be of the type
generally known as a "pseudo" reference electrode which
relies upon the large excess of the oxidizing species to
establish a known potential at a noble metal electrode, in
a preferred embodiment, two electrodes of the same noble
metal are used, however one is generally Of greater surface
area and is used as the reference electrode. The large
excess of the oxidized species and the larger surface area
of the reference resists a shift of the potential of the
reference electrode.

Indicator or working electrode 26 can be either a strip
of platinum, gold, palladium or metallized plastic
positioned on reference electrode 24 or alternately the
working electrode 26 and the reference electrode may be
manufactured as a coplanar unit with electrode 26 being
sandwiched between coplanar electrode 24 material.
Preferable, sample cell 20 is prepared by sandwiching or
laminating the electrodes between the substrate to form a
composite unit.

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15

As shown in Figure 2, first substrate 22 is of a
slightly shorter length so as to expose and end portion 27
of electrodes 24 and 26 and allow for ^.electrical contact
with the testing circuit contained in the apparatus . In
this embodiment, after a sample has been positioned within
well 21, cell 20 is pushed into window 19 of the front panel
to initiate testing. In this embodiment, a reagent may be
applied to well 21, or, preferably, a pad of dry reagent is
positioned therein and a sample (drop) of blood is placed
into the well 21 containing the reagent.

Referring to Figures 4-6, alternative embodiments of
sample cell 20 are shown. In Figure 4, sample cell 120 is
shown having first 122 and second 123 substrates. Reference
electrode 124 and working electrode 126 are laminated
between substrates 122 and 123. Opening 121 is dimensioned
to contain the sample for testing. End 130 is designed to
be inserted into the apparatus, and electrical contact is
made with the respective electrodes through cut-outs 131 and
132 on the cell. Reference electrode 124 also includes cut
out 133 to permit electrical contact with working electrode,.
126.

In Figure 6, working electrode 226 is folded, thereby
providing increased surface area around opening 221, to
achieve increased sensitivity or specificity. In this case,
reference electrode 224 is positioned beneath working
electrode 226. Working electrode includes cut out 234 to

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16

permit electrical contact with reference electrode 224
through cut out 231 in substrate 222. End 230 of substrate
222 also includes cut out 232 to permit electrical contact
with working electrode 226.
5 The sample cell according to the present invention is

positioned through window 19 to initiate the testing
procedure, once inserted, a potential is applied at portion
27 of the sample cell across electrodes 24 and 26 to detect
the presence of the sample. Once the sample's presence is

10 detected, the potential is removed and the incubation period
initiated. Optionally during this period, a vibrator means
31 may be activated to provide agitation of the reagents in
order to enhance dissolution (an incubation period of 20 to
45 seconds is conveniently used for the determination of

15 glucose and no vibration is normally required) . An
electrical potential is next applied at portion 27 of the
sample cell to electrodes 24 and 26 and the current through
the sample is measured and displayed on display 16^

To fully take advantage of the above apparatus, the

20 needed chemistry for the self testing systems is
incorporated into a dry reagent layer that is positioned
onto the disposable cell creating a complete sensor for the
intended analyte. The disposable electrochemical cell is
constructed by the lamination of metallized plastics and

25 nonconducting materials in such a way that there is a
precisely defined working electrode area. The reagent layer

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17

is either directly coated onto the cell or preferably
incorporated (coated) into a supporting matrix such as
filter paper, membrane filter, woven fabric or non-woven
fabric, which is then placed into the cell. When a
5 supporting matrix is used, it pore size and void volume can
be adjusted to provide the desired precision and mechanical
support. In general, membrane filters or nonwoven fabrics
provide the best materials for the reagent layer support.
Pore sizes of 0,45 to 50um and void volumes of 50-90% are

10 appropriate. The coating formulation generally includes a
binder such as gelatin, carrageenan, methylcellulose,
polyvinyl alcohol, polyvinylpyrrolidone, etc., that acts to
delay the dissolution of the reagents until the reagent
layer has adsorbed most of the fluid from the sample. The

15 concentration of the binder is generally on the order of o.l
to 10% with 1-4% preferred.

The reagent layer imbibes a fixed amount of the sample
fluid when it is applied to the surface of the layer thus
eliminating any need for premeasurement of sample volume.

20 Furthermore, by virtue of measuring current flow rather than
reflected light, there is no need to remove the blood from
the surface of the reagent layer prior to measurement as
there is with reflectance spectroscopy systems. While the
fluid sample could be applied directly to the surface of the

25 reagent layer, to facilitate spread of blood across the
entire surface of the reagent layer the sensor preferably

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18

includes a dispersing spreading or wicking layer. This
layer, generally a non-woven fabric or adsorbant paper, is
positioned over the reagent layer and acts to rapidly
distribute the blood over the reagent layer. in some
5 applications this dispersing layer could incorporate
additional reagents.

For glucose determination, cells utilizing the coplanar
design were constructed having the reagent layer containing
the following formulations:
10 Glucose oxidase 600u/ml

Potassium Ferricyanide 0.4M
Phosphate Buffer 0.1M
Potassium Chloride 0.5M
Gelatin 2.0g/dl
15 This was produced by coating a membrane filter with a
solution of the above composition and air drying. The
reagent layer was then cut into strips that just fit the
window opening of the cells and these strips were placed
over the electrodes exposed within the windows. A wicking
20 layer of a non-woven rayon fabric was then placed over this
reagent layer and held in place with an overlay tape.

In order to prove the application of the technology
according to the present, invention, a large number of
examples were run in agueous solution at 25°C. The
25 electrolyte consisted of a phosphate buffer of pH 6.8 which
was about 0.1 molar total phosphate and 0.5 M potassium

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19

chloride reagent. The potentials are referenced to a normal
hydrogen electrode (NHE) . In these tests it was found that
any potential between approximately +0.8 and 1.2 volt (vs
NHE) is suitable for the quantification of hydroquinone when
benzoquinone is used as the oxidant. The limiting currents
are proportional to hydroquinone concentrations in the range
between 0.0001 M and 0.050 M.

Determination of glucose by Cottrell current (i t )
microchronoamperometry with the present method is created in
the reaction of hydroquinone to benzoquinone. Cottrell
currents decay with time in accordance with the equation:

i t . t 1/2 = const

The main difference between these two techniques
consists . of applying the appropriate controlled potential
after the glucose-benzoquinone reaction is complete and
correlating glucose concentrations with Cottrell currents
measured at a fixed time thereafter. The current-time
readout is shown in Figure 8 . Proportionality between
glucose concentrations and Cottrell currents (recorded at t
= 30 -seconds after the application of potential) is shown in
Figure 7.

It should be noted that Cottrell chronoamperometry of
metabolites needs the dual safeguards of enzymatic catalysis
and controlled potential electrolysis. Gluconic acid yields
of 99.9+ percent were attained in the presence of glucose
oxidase. Concomitantly, equivalent amounts of benzoquinone

10

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20

were reduced to hydroquinone, which was conveniently
quantitated in quiescent solutions, at stationary palladium
thin film anodes or sample cells.

The results of these many tests demonstrates the
microchronoamperometric methodology of the present invention
and its practical for glucose self -monitoring by diabetics.

In a presently preferred embodiment of the invention
utilizing ferrocyanide, a number of tests were run showing
certain improved operating capabilities.

Referring to Figure 9, a schematic diagram of a
preferred circuit 15 for use in the apparatus 10 is shown.
Circuit 15 includes a microprocessor and LCD panel 16 . The
working and reference electrodes on the sample cell 20 make
contact at contacts W (working electrode) and R (reference
15 electrode) , respectively. Voltage reference 41 is connected
to battery 42 through analogue power switch 43. Current
from the electrodes W and R is converted by adjustable
resistor 44, and voltage to frequency converter 46
electrically connected to the microprocessor. Other
circuits within the skills of a practiced engineer can
obviously be utilized to obtain the advantages of the
present invention. -

With regard to Figure 10, cell 400 consists of coplanar
working 426 and reference 424 electrodes laminated between
an upper 422 and lower 426 nonconducting material.
Lamination is on an adhesive layer 425. The upper material

20

25

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21

422 includes a die cut opening 428 which , along with the
width of the working electrode material defines the working
electrode area and provides (with an overlapping reagent
layer not depicted) the sampling port of the cell. At one
end of cell 400 is an open area 427 similar to end position
27.

The efficiency of using the apparatus according to the
present invention to provide a means for in-home self
testing by patients such as diabetics (in the preferred
embodiment) can be seen in the following table in which the
technology according to the present invention is compared to
four commercially available units. As will be seen, the
present invention is simpler, and in this instance
simplicity breeds consistency in results.

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GLUCOSE SYSTEM CQSgARISONS

Steps 2

Turn Instrument On x

Calibrate Instrument x

Finger Puncture x

Apply Blood x

Initiate Timing

Sequence "* x

Blot x

Insert Strip to Read X

Read Results x

Total Steps Per

Testing 8

Detection System rs*

15%
10%
5%

X
X
X
X

x

X
X
-JL.

a

RS

3
X

X
X

X
X
X

7

RS

Present
Inven-
-4 tion

15%
10%
5%

Range (mg/dl)

CV** Hypoglycemic
Euglycemic
Hyperglycemic

Correlation 0.921 0.862

(*RS - Reflectance Spectroscopy)

**Coefficient of variation

X
X

X

5
RS

10-400 40-400 25-450 40-400

X
X

Polar o-
graphic

0-1000

5%
3%
2%

0.95

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23

With specific regard to the determination of cholesterol
utilizing the present invention, the generalized chemistry
may be depicted as:
Scheme I

Cholesterol Esters + HjO + CE > Cholesterol + Fatty acid (1)

Cholesterol + OX +CO > Cholestenone + Red (2)

Red > Ox + e- (3)

where the enzymes cholesterol esterase (CE) and cholesterol
oxidase (CO) catalyze reactions 1 and 2 respectively and CO
permits electron transfer with a variety of electroactive
couples (Ox and Red) . Reaction 2 is novel in that electron
acceptors other than dioxygen may be used to oxidize
cholesterol in the presence of the enzyme cholesterol
oxidase. Reaction 1 is well known to those in the field and
is necessary for the determination of total cholesterol
(free cholesterol and cholesterol esters) . Reaction 3 is an
electro-oxidation process for probing and quantitating the
cholesterol .

Utilizing alternative oxidants according to the present
invention, the specific reactions become:
A:

Reaction 1 above

Cholesterol + 2Perricyanide -CO > Cholestenone + 2Ferrocyanide

Ferrocyanide > Ferricyanide + le-

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24

B:

Reaction 1 above

Cholesterol + Benzoquinone -CO > Cholestenone + Hydroquinone

Hydroquinone > Benzoquinone + 2H+ + 2e-

Cholesterol oxidase (CO) from a variety of sources will
catalyze electron transfer from cholesterol to a variety of
the oxidants including benzoquinone, benzoquinone
derivatives such as methylbenzoquinone, ethylbenzoquinone,
chlbrobenzoquinone, ortho-benzoquinone (oxidized form of
catechol), benzoquinonesulfonate, and potassium
ferricyanide. it is also anticipated that the enzyme will
allow electron transfer with other alternate oxidants. As
indicated in Reaction 3 r the reduced product can then be
monitored amperometrically for the quantitative
determination of cholesterol.

Sources of the enzyme catalyzing the oxidation of
cholesterol with alternate oxidants include CO from
Nocardia, Streptomyces, Schizophyllum, Pseudomanas, and
Brevibacterium; experimental conditions under which it is
able to rapidly catalyze the oxidation of cholesterol by
benzoquinone or any of the other oxidants depend somewhat
upon the source of the enzyme. For example, CO from
Streptomyces rapidly catalyzes substrate oxidation with
benzoquinone in phosphate buffer in the presence of any of
a variety of the surfactants including
octylgluconopyranoside and CHAPSO; the same reaction under

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25

Identical conditions with CO from either Brevibacterium or
Nocardia is slower. However, both Nocardia and

Brevibacterium sources are active catalysts for cholesterol
oxidation by alternate oxidants under other conditions.
5 The oxidant also plays a role in which the enzyme is

most active. For example, cholesterol oxidase from Nocardia
rapidly catalyzes substrate oxidation with benzoquinone in
0.2 molar TRIS buffer and 3 g/dL CHAPSO but is slower with
ferricyanide under identical conditions; the Brevibacterium
10 source of the enzyme is relatively inactive with
ferricyanide in TRIS buffer with a variety of surfactants
but when benzoquinone is used as the oxidant the reaction is
very fast. Alternatively, the Schizophyllum source of the
enzyme CO rapidly catalyzes the oxidation of cholesterol in
15 phosphate buffer with either ferricyanide or benzoquinone
and with a variety of surfactants as activators .

As indicated, cholesterol oxidase will catalyze the
oxidation of cholesterol by ferricyanide. Additional
examples where CO catalyzes cholesterol oxidation by
20 ferricyanide include a Nocardia source in TRIS buffer with
a variety of surfactants including sodium deoxycholate,
sodium taurodeoxycholate, CHAPS, Thesit, and CHAPSO.
Furthermore, CO from Nocardia will also catalyze substrate
oxidation with ferricyanide in phosphate buffer with sodium
25 dioctylsulf osuccinate , sodium deoxycholate , sodium
taurodeoxycholate, and Triton X-100. The buffer

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26

concentration is from o.l to 0.4 molar. Surfactant
concentration for maximum activity of the oxidase enzyme
varies with each detergent. For example, with deoxycholate
or taurodeoxycholate, the enzyme in 0.2 M TRIS is most
active with detergent in the range from 20 to 90 millimolar.
However, enzyme catalytic activity is observed up to and
through a 10% concentration, with octyl-gluconopyranoside,
the maximum activity of the enzyme with the oxidant
ferricyanide occurs at a detergent concentration of
approximately 1.2%; however, the enzyme still maintains
activity at higher and lower concentrations of the
surfactant.

Both esterase and CO require a surfactant for high
activity, specific surfactants include sodium deoxycholate,
sodium taurodeoxycholate, sodium glycodeoxycholate, CHAPS
( 3-(3-chlolamidopropyl) dimethylammonio-l-propanesulf onate) ,
CHAPSO (3-(3-chlolamidopropyl) dimethylammonio-2-hydroxy-l-
propanesulf onate) , octyl-gluconopyranoside, octyl-
thiogluconopyranoside, nonyl-gluconopyranside, dodecyl-
gluconopyranoside, Triton X-100, Dioctyl sulfosuccinate,
Thesit (Hydroxypolyethoxydodecane) , and lecithin
(phosphatidylcholine) . Buffers acceptable for this reaction
to occur with the enzyme include phosphate, TRIS, MOPS, MES,
HEPES, Tricine, Bicine, ACES, CAPS, and TAPS. An alternate
generallized reaction scheme f P r the measurement of
cholesterol in serum and other biological fluids is given

.0

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27

Scheme II

Cholesterol Esters -CE > Cholesterol + Patty Acid i

Cholesterol + Ox x -CO > Cholestenone + Redj 4

Redj + Ox 2 > oxj + Red 2 5

Red 2 > Ox 2 + e- - 6

where Ox, and Red 2 function as an electron mediator couple
between the cholesterol and the electroactive couple
Ox 2 /Red 2 . In this case, Ox 1 and Red 1 need not be
electroactive because they do not have to participate in the
electrooxidation process (Reaction 6). However, from both
a thermodynamic and kinetic perspective, this couple with
the assistance of the enzyme cholesterol oxidase must be
able to accept electrons from cholesterol and relay them to
the electroactive couple (OXj/Redj) .
> Specific examples of this chemistry include
Example 1

Reaction 1 above

Cholesterol + Benzoquinone -CO > Cholestenone + Hydroquinone

Hydroquinone + 2Ferricyanide — > Benzoquinone + 2Ferrocyanide
Ferrocyanide > Ferricyanide + le-

Schfeme II is. beneficial when the rate of reaction of
cholesterol with the electroactive oxidant as in Scheme I is
so slow that it precludes its use in a practical sensor. As
mentioned above, Scheme II is also beneficial when the

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28

electron mediator itself (o^/Red,) is either not
electroactive or exhibits poor electrochemistry under
conditions of the „enzyme chemistry. it is under these
conditions that Scheme II is particularly applicable, other
electron mediators (O^/Red,) between cholesterol and
f erricyanide for use in Scheme II may be possible including
Phenazine ethosulfate, phenazine methosulf ate,
tetramethyibenzidine, derivatives of benzoquinone,'
naphthoquinone and naphthoquinone derivatives, anthraquinone
and anthraquinone derivatives, catechol, phenylenediamine,
tetramethylphenenediamine, and other derivatives of
phenylenediamine.

Furthermore, while it is understood that the oxidized
form of the electron relay accepts electrons from
cholesterol, in the sensor either the oxidized or the
reduced form of the mediator may be incorporated provided it
reacts rapidly with both cholesterol and ferricyanide. if
the reduced form is sufficiently stable and the oxidized
form is not, then reductant, may be incorporated into the
sensor in relatively small quantity (in comparison with the
analyte to be determined) and still provide the electron
relay. However, this causes a corresponding background
signal that must be accounted for. The reductant, must also
be isolated from ferricyanide in the sensor by incorporation
into a separate reagent layer.

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29

Several formulations of the above chemistries
encompassing both Schemes I and II have been prepared as dry-
films on . membranes . These membranes are positioned in the
sensor which can then be used for the determination of
cholesterol. A preferred formulation of the reagents
involving Scheme II consists of the following
Cholesterol Esterase § 400 Units/mL

Cholesterol Oxidase from Streptomyces @ 200 Units/mL

0.05 molar Potassium Ferricyanide

0.5 molar Potassium Chloride

0.2 molar Phosphate, pH 6.9

3 g/dL CHAPSO

2 g/dL gelatin

and 0.0001 molar hydroquinone (in the spreading or wicking
layer) .

The concentrations provided are that of the solutions which
are coated onto porous supports, filter paper or membranes;
these concentrations are reestablished when the membrane
imbibes the serum or whole blood specimen. For cholesterol
determinations larger pore sizes in the filter support are
necessary than that used for glucose. This is because the
cholesterol resides in the serum in large lipoproteins
(chylomicrons, LDL, VLDL-, and HDL) which must penetrate the
various layers of the sensor until they reach the reagents.
The surfactants to a major extent break these natural
micelles up into smaller micelles providing a greater total

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30

surface area on which the enzymes catalyze the reaction.
Due to the instability of benzoquinone a small quantity of
hydroquinone, which is more stable by nature of its lower
vapor pressure, is. incorporated into the sensor to assist
electron mediation between cholesterol and ferricyanide.
Upon introduction of the serum specimen into the sensor the
hydroquinone is oxidized to benzoquinone; the benzoquinone
is then free to pick up electrons from the substrate and
cycle them to ferricyanide. Under these conditions the rate
of the reaction of cholesterol with a small quantity of
benzoquinone is more rapid than that with a large excess of
ferricyanide.

An alternate and preferred formulation of reagents
utilizing scheme II that may be incorporated into the
reagent layer of the sensor is:

Cholesterol Oxidase from Streptomyces @ 200 Units/mL

Lipase from Candida § 500 Units/mL

3 g/dL CHAPSO

0.2 molar TRIS, pH 7.5

0.05 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
0.05 Molar MgCl 2
2 g/dL gelatin

and .0.001 molar hydroquinone (in the spreading layer) .
The magnesium salt in this formulation increases stability
of the esterase enzyme in the phosphate-free reagent layer;

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31

Lipase assists the break up of the lipoproteins. With these
dry reagent layers incorporated into the sensor and using
the evaluation methodology as described, the following
results were obtained.
5 Serum Cholesterol, ma% Average Current, uA

91 . 19.3
182 27.2
309 38.5
These results demonstrate the quantitative response of
10 the sensor to serum cholesterol levels.

Alternate and preferred embodiment of the sensor
utilizing Scheme I is provided by reagent compositions:
Cholesterol Esterase § 400 Units/mL
Cholesterol Oxidase from Nocardia @ 200 Units/mL
15 1 g/dL Triton X-100

0.1 molar TRIS buffer, pH 8.6
0.2 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
0.02 molar MgCl 2
20 2 g/dL gelatin

OR . ..

Cholesterol Esterase § 200 Units/mL

Cholesterol Oxidase from Streptomyces @ 200 Units/mL
0.06 molar Sodium deoxycholate
25 0.1 molar TRIS buffer, pH 8.6

0.2 molar Potassium Ferricyanide

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0.5 molar Potassium Chloride
2 g/dL gelatin

Thus, while we have illustrated and described the
preferred embodiment of my invention, it is to be understood
that this invention is capable of variation and
modification, and we therefore do not wish or intend to be
limited to the precise terms set forth, but desire and
intend to avail ourselves of such changes and alterations
which may be made for adapting the invention of the present :
invention to various usages and conditions. Accordingly,
such changes and alterations are properly intended to be
within the full range of equivalents, and therefore within
the purview, of the following claims. The terms and
expressions which have been employed in the foregoing
specifications are used therein as terms of description and
not of limitation, and thus there is no intention, in the
use of such terms and expressions, of excluding equivalents
of the features shown and described or portions thereof, it
being recognized that the scope of the invention is defined
and limited only by the claims which follow.

Having thus described our invention and the manner and
process of making and using it in such full, ci ear , concis6r
and exact terms so as to enable any person skilled in the

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33

art to which it pertains, or to with which it is most nearly
connected , to make and use the same.

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34

WE CLATM

■ 1. A method for measuring the amount of a selected compound
in body fluids comprising

5 a * Plying a sample of fluid to be tested in a

sample cell having first and second electrodes;

b. mixing said sample with an oxidant and a
buffer;

c. applying a potential across said electrodes
10 and sample; and

d. measuring the resultant current to determine
the concentration of said select compound present in
said sample.

15 2. A method as set forth in claim 1, wherein the

compound is selected from the group consisting of glucose,
cholesterol, TSH, : T4, hormones, antiarrhythmics,'
antiepiieptics and nontherapeutic drugs.

2 ° 3. a method as set forth in Claim 1, wherein the

oxidant is selected from the group consisting of
benzoguinone, ferricyanide, ferricinium, cobalt (Hi) , tris
orthophenantroline, and cobalt (ui) tridipyr idyl .

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35

4. A method as set forth in Claim 1 wherein the
electrodes of the sample cell comprise a reference and a
working electrode.

5. A sample cell comprising

a. first and second nonconductive substrates,
said first substrate having a opening therethrough;

b. a metallized first electrode positioned on one
of said second substrate; and

c. a second electrode positioned on said first
electrode, whereby said first electrode is positioned
over a portion of said second electrode to form a
laminate with said first and second electrodes
positioned therebetween and said opening exposing said
electrodes to define a sample well.

6. A sample cell as set forth in Claim 5, wherein
first electrode comprises a reference electrode and said
second electrode is a working electrode.

7. A sample cell comprising first and second
nonconducting substrates and first and second metallized
electrodes, said first electrode being .positioned on said
second substrate and said second electrode including a

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36

nonconducting substrate interpositioned between said first
and second substrates; an opening through said first
substrate and said second electrode, said opening defining
a sample well.

8. A sample cell as set forth in Claim 7, wherein said
first electrode is a working electrode and said second
electrode is a reference electrode and wherein said opening
in the second electrode is smaller than said opening of the
first substrate.

9. A sample cell as set forth in Claim 7, wherein said
first electrode is a reference electrode and said second
electrode is a working electrode.

10. A sample cell comprising first and second
nonconducting substrates and a working end reference
electrode, said reference electrode comprising a metallized
layer on said second substrate and said working electrode
comprising a metallized layer on a nonconducting support
laminated between said- first and second substrates, said
working electrode including a convoluted portion with an
opening therethrough defining a supple well, and an opening

WO 89/08713

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37

through said first substrate aligned with said opening in
said working electrode,

11. A laminated sample cell as set forth in Claim 10,
5 wherein said first substrate includes a pair of notches
exposing and defining, a contact area on said working
electrode and a contact area on said reference electrode and
a notch in said working electrode positioned under said
notch in said first substrate to define said reference
10 electrode contact area.

12 • An apparatus for measuring blood glucose comprising

a. a housing;

b. circuit board mounted within said housing
15 having thereon electrical contacts adapted to

electrically contact the electrodes of a sample cell
containing a sample to be measures;

c a sample cell containing first and second
electrodes and a well for containing a sample to be
20 tested;

d. an opening in said housing to permit a sample
cell to be inserted into said housing and contact said
electrical contact;

WO 89/08713

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38

e. means for applying an electrical potential to
said electrodes; and

t. means for measuring the electrical current
tRrough said sample •

13. Apparatus set forth in Claim 12 wherein said means
for measuring electrical current through said sample
includes a microprocessor.

14. Apparatus as set forth in Claim 12 wherein said
window includes means for initiating the vibration of sample
cell and electrical potential upon insertion of said sample
cell.

15. Apparatus as set forth in Claim 12 wherein said
electrodes of said sample cell comprise a reference and
working electrode „

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1/6

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3/6

SUBSTITUTE SHEET

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4/6

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SUBSTITUTE SHEET

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6/6

PCT/US89/01057

INTERNATIONAL SEARCH REPORT

International AoDhcarion No.

PCT/US89/Q1057

I, CLASSIFICATION OF SUBJECT MATTER [>t several Classification symbols apply, .r.gicate 3II1 *
Accoro»ng 10 Internat.onai Patent Classification OPC) or to ooth National Classification anc IPC

IPC U): C12Q 1/00, 1/26? C12M 1/34. G01N 27/26
P-S. Cln 415/4, 2?, 291; 204/403

II. FIELDS SEARCHED

Minimum Documentation Searched '

Classification System

Classification Symbols

U.S.

435/4, 11, H, 25, 288, 291, 817
204/403, 415

436/63, 150. 151. 817: A22/68. 78. Q8

Documentation Searched other than Minimum Documentation
to the Extent that such Ooeuments are Included in the Fields Searched *

III. OOCUMENTS CONSIDERED TO BE RELEVANT »

Category '

Citation of Document. 11 with indication, where appropriate, of the relevant passages a

Relevant to Claim No. * J

X
X

I
I

US, A, 4,005,002 (RACINE ET AL) 25 January 1977,
See column 1, line 46 to column 2, line 25,
column 3, lines 5 to 20, column 6, lines 15
to 33 and column 8, lines 16 to 17.

US, A, 3,838,033 (MJJDT ET AL) 24 September 1974,
See column 2, line 52 to column 3, line 19
and column 3, lines 46 to 75.

US, A, 4,225,410 (PACE) 30 September 1980,
See the entire document.

US, A, 4,217,196 (HtJCH) 12 August 1980,

See column 3, line 35 to column 4, line 45.

US, A, 3,925,183 (0SWIN ET AL) 09 December 1975,
See column 2, lines 14 to 24 and column 5,
line 28 to column 6, line 53.

US, A, 4,169,779 (TATABIA ET AL) 02 October 1979,
See column 3, line 18 to column 4, line 30*

1-4

1-4

5-15
5-15
5-11

5-11

* Special categories of cited documents: w
"A" document defining the general state of the art which is not
considered to be of particular relevance

"E** earlier document but pubNshed on or after the international
filing date

"L~ document which may throw doubts on priority ctaim fs) or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O* document referring to an oral disclosure, use. eihibition or

"P" document published prior to the international fifing date but
later than the priority date claimed

"V later document published after the international filing date
or priority date and not In conflict with the application but
ated to understand the principle or theory underlying the
invention

-X* document of particular relevance: the claimed invention
cannot be considered novel or cannot oe considered to
Involve an inventive step

"Y" document of -particular relevance: the claimed invention
cannot be considered to Involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art

document member of the same patent family

IV. CERTIFICATION

Date of the Actual Completion of the International Search

05 June 1989

. Date of Mailing oUbL

2 6 Jfll

^nte^e|0|ej Search Report

International Searching Authority

Sign^^^o^A^thjpj

ISA/US

Randall E.

Dec*

Form PCTftSAOlO (moorO meat) {fwv.n-87)

International Application No.

PCT/US89/01057

FURTHER INFORMATION CONTINUEP FROM THE SECOND SHEET

US, A, 4-,682,602 (EROH&SKA) 28 July 1987

See column 2, lines 33 to 62 and column 3,
lines 24 to 59.

V Q OBSERVATIONS WHERE CERTAIN CLAIMS WERE

5-11

FOUND UNSEARCHABLE'

This int.mat.ona. search report has not been established m respect of certain claim, under Article UO) (a) tor the following „,„„, .
Claim number. . because they relate to subject matter " not required to be searched by this Authority, namely:

10 r;r w ^snrsrs:s

3-D Claim numbers
PCT Rule 6.4(a).

, became they am dependent claims not drafted In accordance with the second and third sentences of

VL D OBSEWATtOHS WHERE UNITY OF INVENTION IS LACKING*

This International Searching Authority found multiple inventions In

this international application as follows:

, ' D of'tn. ^lo^apSf "** ^ * aMieantl * h " ™* "Pot "vers ,« tenable cfalm.

lD --^1h^^^^^ — — — — on.

X D No required additional search fees ^
the invention first mentioned in the

timely paid by the applicant Consequently, this International search report is restricted to
wt ft** covered by claim numbers:

* ° ^l^^^mlnnZ™*" 1 ^ 0 * eff0rt "» internatiomd S.archmg Authority did not

on Protest

□ The additional search lees were accompanied by applicant's protest.

□ No protest accompanied the payment of additional search fees.

fcmPCT/tSH&Q

*B(Rnr. 11-87)